CD8+ T-cell mediated immunity can eliminate large tumor burdens. However, significant clinical responses of cancer patients and mice with established vascularized tumors are difficult to achieve with most vaccination strategies. Insufficient trafficking and activation of immune cells at tumor sites are thought to be, at least in part, responsible for the failure of immunotherapy (IT) to destroy solid tumors. Ionizing radiation therapy (RT) is an important local cancer treatment. Used as cytocidal agent, RT can also alter the tumor microenvironment and generate inflammation. In the 4T1 mouse model of metastatic breast cancer we have shown that RT in combination with CTLA-4 blockade elicits a CD8+ T cell-mediated response inhibiting metastases and inducing regression of primary tumors. Accumulated data support the hypothesis that RT can be used as an immunological adjuvant to enhance the effectiveness of IT. However, the ability of ionizing radiation to promote anti-tumor immunity is still controversial, and mechanisms involved remain largely undefined. Proposed studies will determine the mechanismswhereby RT promotes anti-tumor immunity by analyzing the critical steps of this process. First, the ability of RT to promote cross-priming will be determined using the 4T1 tumor model and its derivative expressing a reporter antigen. Presentation of tumor antigens by dendritic cells and activation, dissemination and persistence of tumor-specific T cells will be analyzed. Second, the effect of RT on effector T cell trafficking and function within tumors will be determined. The role of CXCR6/CXCL16 interactions in CD8+ T cell recruitment to irradiated tumors will be established employing EGFP-knockin in the CXCR6 locus mice, and RNA interference to silence CXCL16. In addition, intravital microscopy will be used to study the dynamic behavior of CXCR6+ T cells in the microenvironment of established 4T1 tumors and their response to local radiation. Third, the ability of RT to induce anti-tumor responses in combination with another immune stimulatory antibody (anti-4-1BB/CD137) will be tested and compared to CTLA-4 blockade. Findings will then be confirmed in a transgenic mouse model of spontaneous carcinogenesis. The innovative concept that RT can be used as an immunological adjuvant to enhance the effectiveness of IT will open a new field of investigation. Overall, these studies will provide the pre-clinical data necessary for testing this novel therapeutic strategy in future clinical trials.